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<em>Pseudomonas aeruginosa</em> is Present in Crude Oil Contaminated Sites of Barmer Region (India) | OMICS International
ISSN: 2155-6199
Journal of Bioremediation & Biodegradation

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Pseudomonas aeruginosa is Present in Crude Oil Contaminated Sites of Barmer Region (India)

Bharti Prakash and Mohammad Irfan*
Department of Zoology, Government College, Ajmer (Rajasthan) India
Corresponding Author : Mohammad Irfan
Department of Zoology
Government College
Ajmer (Rajasthan), India
Tel: +91-98290 69078
E-mail: [email protected] yahoo.com
Received June 26, 2011; Accepted November 03, 2011; Published November 06, 2011
Citation: Prakash B, Irfan M (2011) Pseudomonas aeruginosa is Present in Crude Oil Contaminated Sites of Barmer Region (India). J Bioremed Biodegrad 2:129. doi:10.4172/2155-6199.1000129
Copyright: © 2011 Prakash B, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract

Purpose: Refinery effluent and oil spills are the major sources of oil pollution. Diverse microbial population including Bacteria, Fungi and Algae can metabolize the hydrocarbons found in crude oil. Among the microorganisms bacteria are usually the choice. The aims of present study are

• Isolation of Pseudomonas aeruginosa from crude oil contaminated sites of Barmer Region (India)

• Identification of Pseudomonas aeruginosa on the basis of its specific characteristics

Methods: Oil contaminated soil samples were collected randomly from five different sites of Mangala oil field in Barmer district (India), where huge amount of crude oil has been discovered by Cairns India Energy Company. Isolation was carried out by serial dilution agar platting method at 37˚C using Bushnell-Haas agar medium+crude oil as selective medium.

• Results: The Pseudomonas aeruginosa colonies were identified by a combination of information from primary and secondary identification. Morphological, physiological and biochemical characteristics of pure isolates revealed that Gram-negative rods isolate were Pseudomonas aeruginosa.

• Conclusions: Previous observations have identified the Pseudomonas genus most efficient among hydrocarbon degrading microorganisms. Further, the use of surfactants specially, rhamnolipids has been found to enhance degradation of crude oil. Pseudomonas aeruginosa is a typical strain for rhamnolipid production and can utilize crude oil as the sole carbon source. Data from this study showed the presence of Pseudomonas aeruginosa in the oil contaminated soil and lend weight to this suggestion that Pseudomonas aeruginosa exhibit oil degrading capabilities.

Keywords
Barmer district (India); Crude oil contaminated soil; Pseudomonas aeruginosa
Introduction
Petroleum hydrocarbon continues to be used as the principle source of energy and hence resulted in an important global environmental pollutant. Refinery effluent and oil spills are the major sources of oil pollution. In India 20 refineries and 10 different operating companies are discharging effluents at an alarming rate. These sources play a major role in polluting the environment and the abutting landscape. This severely affects ecosystem. With the focus on the protection of environment and pollution control in India, most of refineries with conventional acid clay process are likely to face serious threat in coming years and need has been felt for a safe, environmental friendly process which eliminates the use of acid and at the same time suitable for smaller capacities since collection of large quantities of used oil is a limitation in our country.
At present, bioremediation (use of microorganisms to remove pollutants) is often the most suitable method for remediation of especially petroleum hydrocarbons, because it is cost effective and it converts the petroleum hydrocarbons into the harmless by-products such as carbon dioxide and water.
Diverse microbial population including Bacteria, Fungi and Algae can metabolize the hydrocarbons found in crude oil. Among the microorganisms bacteria are usually the choice because:
- They have more rapid metabolic rates.
- Numerous metabolic pathways of various organic pollutants have been determined in bacteria.
- Bacteria can be genetically manipulated to improve their bioremediation capabilities.
There are at least 22 genera of bacteria that can metabolize petroleum hydrocarbons which include- Pseudomonas, Aeromonas, Bacillus, Flavobacterium, Corynebacterium, Micrococcus etc. Based on crude oil degradation capacity Pseudomonas aeruginosa is the most active hydrocarbon utilizer in crude oil. Previous observations have identified the Pseudomonas genus most efficient among hydrocarbon degrading microorganisms [1-3].
Further the use of surfactants has been found to enhance degradation of crude oil [4,5]. Among various surfactants, rhamnolipids are considered to be the most in degrading hydrocarbons [6].
Pseudomonas aeruginosa is a typical strain for rhamnolipid production and can utilize crude oil as the sole carbon source. The Pseudomonas aeruginosa also able to tolerate and grow in high concentrations (up to 50% v/v) of crude oil and able to utilize compounds such as aliphatic and monoaromatic hydrocarbons as well as alcohols as substrates.
Materials and Methods
Sampling
In the present study, oil contaminated soil samples were collected randomly from five different sites of Mangala oil field in Barmer district, Rajasthan where huge amount of crude oil has been discovered by Cairns India Energy Company.
These samples were collected in an autoclaved vial by using simple sampling technique. Care was taken in handling and sampling to avoid contamination of the samples and returned to the laboratory for bacterial extraction as soon as possible. If necessary, the sample vials were held under refrigeration at 4˚C until extraction, which was no later than 48 hours after sampling.
Isolation of bacteria
Crude oil was sterilized by autoclaving at 121˚C for 15 minutes in a sealed glass test tube. The crude oil acted as a source of carbon for the oil degrading bacteria.
Bushnell-Haas medium was used for the isolation of hydrocarbon degrading bacteria. Bushnell-Haas medium was prepared by adding 1g KH2PO4, 1g K2HPO4, 1g NH4NO3, 0.3g Cholesterol, 0.2g MgSO4.7H2O, 0.05g FeCl3, 0.02g CaCl2.2H2O and finally 15g/L of agar were added; 1000ml of water was added to the flask containing Bushnell-Haas medium. Since the media had a total volume of more than 1000ml hence 700ml of this mixture was transferred to a separate 1 liter flask to avoid spill over during autoclaving. The Bushnell-Haas medium was then autoclaved at 121˚C for 15 minutes.
The sub samples of 1g soil were accurately weighed before being transferred aseptically to a bottle containing 99ml of sterile dilute saline solution to attain a dilution of 10-2. The mixture was then shaken vigorously and was allowed for the soil to settle at the bottom of the bottle and 0.1ml of the water was transferred to a test tube containing 9ml of sterile dilute saline solution to attain a dilution of 10-3. A serial dilution technique subsequently yielded 4 additional test tubes with dilutions of 10-4, 10-5, 10-6 Using a pipette, 1ml of the solution from each of the test tubes was inoculated in Bushnell-Haas medium containing Petri dishes by platting and spreading method. 0.5ml of the crude oil was then transferred to the Petri dishes. This process selects for organisms that exhibit hydrocarbon degrading capabilities. The Petri dishes were incubated at 37˚C for 24-48 hours.
Identification of bacteria
The Pseudomonas aeruginosa colonies were identified by a combination of information from primary and secondary identification. Morphological, physiological and biochemical characteristics of pure isolates were examined according to the Bergey’s Manual of Determinative Bacteriology.
Primary identification was done on the basis of colony and cell morphology and Gram staining. Representative colonies of Pseudomonas aeruginosa appeared on plates were checked for purity through the microscopy and pure isolates were streaked on slants of Bushnell-Haas medium on which they developed during isolation and stored at 4˚C for further investigation.
After pure cultures had been obtained, four biochemical tests: Casein, Citrate, Urease and MacConkey were performed for secondary identification. Tests were conducted as described by Cappuccino and Sherman [7]. After identification the isolates were deposited in the culture collection for long-term preservation.
Results
Pseudomonas aeruginosa was successfully isolated from crude oil contaminated soil samples using serial dilution agar plating method. This culturing technique based upon the principle that when materialcontaining microorganism is cultured each viable microorganism will develop into a colony [8].
It is known from literature that Pseudomonas aeruginosa colonies are large, opaque, shiny colonies with serrated edges. On the basis of Gram staining and microscopic appearance these colonies had a cell type characteristic of gram-negative rods.
All four of the biochemical test results supported a positive Pseudomonas aeruginosa identification after 48 hours incubation. The casein test appeared to show clearing through the medium, characteristic of a positive casein test. The citrate medium had a color change from green to dark blue, indicative of a positive result for citrate test. The urease agar had a color change from yellow to a bright pink, a positive characteristic for the presence of the enzyme urease. The MacConkey test showed growth, but the colonies were colorless, not red, characteristic of no lactose fermentation, or a negative test result.
Discussion
Data from this study showed the presence of Pseudomonas aeruginosa in the oil contaminated soil and lend weight to this suggestion that Pseudomonas aeruginosa exhibit oil degrading capabilities which can be used for the bioremediation of crude oil polluted sites.
Acknowledgements
The authors are grateful to Department of Zoology, Government College, Ajmer (India) for all facilities provided during the course of the study.
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